Method for effecting angiogenesis by administering nigella sativa

ABSTRACT

A method for effecting angiogenesis in a subject at risk of or experiencing ischemia related to the heart, brain, kidney, intestine, and/or legs. The method involves administering Nigella sativa to the subject. The method may also treat ischemia.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/539,028 filed Jul. 31, 2017, which is incorporated herein byreference in its entirety.

STATEMENT REGARDING PRIOR DISCLOSURE BY THE INVENTOR

Aspects of this technology are described in a conference abstract“Coronary angiogenic effect of long term administration of Nigellasativa” by Lubna Ibrahim Al Asoom, in J Clin Exp Cardiolog, 2016, 7:8,which is incorporated herein by reference in its entirety.

BACKGROUND Field of the Disclosure

This disclosure relates to a method for effecting angiogenesis in asubject in need thereof. The method may treat ischemia related to theheart, brain, kidney, legs, and/or intestine.

Description of the Related Art

Coronary artery disease (CAD) is one of the leading causes of deathworldwide. The World Health Organization (WHO) reports that 31% ofdeaths across the globe are due to cardiovascular diseases, and ischemicheart disease accounts for 46% of deaths from CAD (Alwan A, Armstrong T,Bettcher D, Boerma T, Branca F, Ho J, et al. Global atlas oncardiovascular disease prevention and control Policies, strategies andinterventions. WHO; World Heart Federation; World Stroke Organization,2011, incorporated herein by reference in its entirety). Risk factors ofcoronary artery disease include obesity, hypercholesterolemia,hypertension, diabetes, and smoking (Mack M, Gopal A. Epidemiology,Traditional and Novel Risk Factors in Coronary Artery Disease. HeartFail Clin. 2016 January; 12(1):1-10. PubMed PMID: 26567971, incorporatedherein by reference in its entirety).

Exercise has been recommended as a cardiovascular protective measure.Favorable cardiac and coronary adaptations are well known to result fromexercising, and they have been reported in the literature (Brown M D.Exercise and coronary vascular remodelling in the healthy heart. ExpPhysiol. 2003 September; 88(5):645-58. PubMed PMID: 12955165; andBernardo B C, McMullen J R. Molecular Aspects of Exercise-inducedCardiac Remodeling. Cardiol Clin. 2016 November; 34(4):515-30. PubMedPMID: 27692221, each incorporated herein by reference in theirentirety). Exercise can enhance myocardial blood flow through multiplestructural and physiological alterations of the coronary vascular bed.This includes increase in size of the conduit of large arteries,increases in the number of arterioles and induction of capillary growth(Tomanek R J. Exercise-induced coronary angiogenesis: a review. Med SciSports Exerc. 1994 October; 26(10):1245-51. PubMed PMID: 7528318,incorporated herein by reference in its entirety). A wide range ofstimuli have been suggested to explain the angiogenic effect of exercisetraining. During short bouts of exercise, the increased myocardial bloodflow leads to increased shear stress and the release of endothelialvasodilators and nitric oxide (Zhang J, Friedman M H. Adaptive responseof vascular endothelial cells to an acute increase in shear stressfrequency. Am J Physiol Heart Circ Physiol. 2013 September;305(6):H894-902. PubMed PMID: 23851277. Pubmed Central PMCID:PMC3761335, incorporated herein by reference in its entirety). Growthfactors, such as fibroblast growth factors (FGFs), vascular endothelialgrowth factor (VEGF), and insulin growth factor-I (IGF-I), have beenfound to be upregulated in the coronary circulation in exercise-trainedanimals (Smith S K. Angiogenesis, vascular endothelial growth factor andthe endometrium. Hum Reprod Update. 1998 1998 September-October;4(5):509-19. PubMed PMID: 10027603, incorporated herein by reference inits entirety).

The method of assessing the increase in the coronary circulation inhumans and animals which exercised varies. In exercise-trained humansubjects and athletes, coronary circulation capacity is assessed byevaluating coronary flow, using electromagnetic flowmeter or ultrasonicdevices (Papanastasiou G, Williams M C, Dweck M R, Alam S, Cooper A,Mirsadraee S, et al. Quantitative assessment of myocardial blood flow incoronary artery disease by cardiovascular magnetic resonance: comparisonof Fermi and distributed parameter modeling against invasive methods. JCardiovasc Magn Reson. 2016; 18(1):57. PubMed PMID: 27624746. PubmedCentral PMCID: PMC5022209, incorporated herein by reference in itsentirety). In animals, immunohistochemical studies are used to label thevascular tree and measure the cross-sectional area (Marini M, FalcieriE, Margonato V, Treré D, Lapalombella R, di Tullio S, et al. Partialpersistence of exercise-induced myocardial angiogenesis following 4-weekdetraining in the rat. Histochem Cell Biol. 2008 April; 129(4):479-87.PubMed PMID: 18172661, incorporated herein by reference in itsentirety).

Despite accumulating evidence of the prophylactic influence of exerciseon cardiac health and coronary circulation, the compliance towardexercise is low worldwide. Adjuvant therapeutics aimed to induceangiogenesis are needed either as prophylaxis of ischemia or as anattempt to rescue ischemic tissue.

An objective of the present disclosure is to provide a method forinducing angiogenesis and optionally treat ischemia.

SUMMARY

A first aspect of the disclosure relates to a method for effectingangiogenesis and optionally treating ischemia, the method comprisingadministering a first effective amount of Nigella sativa to a subject inneed thereof.

In one embodiment, the method effects angiogenesis in at least one bodypart of the subject selected from the group consisting of a heart, abrain, and a limb.

In one embodiment, the method treats ischemia which is at least oneselected from the group consisting of brain ischemia, ischemic heartdisease, and limb ischemia.

In one embodiment, the method treats at least one type of ischemic heartdisease selected from the group consisting of stable angina, myocardialinfarction, cardiac syndrome X, and cardiac arrest.

In one embodiment, the method effects angiogenesis in a heart of thesubject and does not lead to cardiac hypertrophy in the subject.

In one embodiment, the first effective amount of Nigella sativa isadministered in a composition comprising water and Nigella sativa seeds.

In one embodiment, the composition comprises 1-50 wt % Nigella sativaseeds, based on a total weight of the composition.

In one embodiment, the Nigella sativa seeds are ground.

In one embodiment, the first effective amount of Nigella sativa isadministered 1-3 times a day.

In one embodiment, the first effective amount of Nigella sativa isadministered for 6-30 weeks.

In one embodiment, the first effective amount of Nigella sativa isadministered 4-6 days per week for 7-10 weeks.

In one embodiment, the first effective amount of Nigella sativa isadministered for 4-6 consecutive days per week.

In one embodiment, the first effective amount of Nigella sativa is in arange of 0.1-1 g/kg body weight of the subject.

In one embodiment, the first effective amount of Nigella sativa is in arange of 0.6-0.9 g/kg body weight of the subject.

In one embodiment, the first effective amount of Nigella sativa isadministered orally.

In one embodiment, the method further comprises measuring a level of atleast one biomarker selected from the group consisting of VEGF, VWF,PECAM-1, and NOS-2 before and/or after the administering of the firsteffective amount.

In one embodiment, the method further comprises administering a secondeffective amount of Nigella sativa.

In one embodiment, the second effective amount of Nigella sativa is in arange of 0.1-1 g/kg body weight of the subject and is different from thefirst effective amount of Nigella sativa.

In one embodiment, the second effective amount of Nigella sativa isadministered for 1 day or 2-10 consecutive days.

In one embodiment, the second effective amount of Nigella sativa isadministered for 4-6 consecutive days.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation and many of the attendant advantagesthereof will be readily obtained as the same becomes better understoodby reference to the following detailed description when considered inconnection with the accompanying drawings, wherein:

FIG. 1A is a photomicrograph of left ventricular tissues (from controlrats) stained with anti-VEGF antibodies.

FIG. 1B is a photomicrograph of left ventricular tissues (from rats fedwith Nigella sativa) stained with anti-VEGF antibodies.

FIG. 1C is a photomicrograph of left ventricular tissues (from ratswhich exercised) stained with anti-VEGF antibodies.

FIG. 1D is a photomicrograph of left ventricular tissues (from controlrats) stained with anti-VWF antibodies.

FIG. 1E is a photomicrograph of left ventricular tissues (from rats fedwith Nigella sativa) stained with anti-VWF antibodies.

FIG. 1F is a photomicrograph of left ventricular tissues (from ratswhich exercised) stained with anti-VWF antibodies.

FIG. 1G is a photomicrograph of left ventricular tissues (from controlrats) stained with anti-PECAM-1 antibodies.

FIG. 1H is a photomicrograph of left ventricular tissues (from rats fedwith Nigella sativa) stained with anti-PECAM-1 antibodies.

FIG. 1I is a photomicrograph of left ventricular tissues (from ratswhich exercised) stained with anti-PECAM-1 antibodies.

FIG. 1J is a photomicrograph of left ventricular tissues (from controlrats) stained with anti-NOS-2 antibodies.

FIG. 1K is a photomicrograph of left ventricular tissues (from rats fedwith Nigella 20 sativa) stained with anti-NOS-2 antibodies.

FIG. 1L is a photomicrograph of left ventricular tissues (from ratswhich exercised) stained with anti-NOS-2 antibodies.

FIG. 2A compares the levels of VEGF between the rats.

FIG. 2B compares the levels of VWF between the rats.

FIG. 2C compares the levels of PECAM-1 between the rats.

FIG. 2D compares the levels of NOS-2 between the rats.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Embodiments of the present disclosure will now be described more fullyhereinafter with reference to the accompanying drawings, in which some,but not all embodiments of the disclosure are shown.

As used herein, the words “a”, “an”, and the like carry the meaning of“one or more”. Within the description of this disclosure, where anumerical limit or range is stated, the endpoints are included unlessstated otherwise. Also, all values and subranges within a numericallimit or range are specifically included as if explicitly written out.

Ischemia is a restriction in blood supply to tissues, causing a shortageof oxygen and glucose needed for cellular metabolism. Ischemia can bepartial (e.g., 20% to less than 100%, 30-80%, or 40-60% of an initialtotal blood supply) or total. Ischemia may be caused by atherosclerosis,which is the deposition and hardening of plaque on the walls of thearteries, thus narrowing or blocking the arteries. The plaque containscholesterol, fat, and calcium.

As oxygen is carried to tissues in the blood, insufficient blood supplycauses tissue to become starved of oxygen. In the highly aerobic tissuesof the heart and brain, irreversible damage to tissues can occur in asquickly as 3-4 minutes at body temperature.

Ischemic heart disease is a condition that affects the supply of bloodto the heart. Ischemic heart disease may be stable angina, unstableangina, myocardial infarction, cardiac syndrome X, or cardiac arrest.

Brain ischemia is a condition in which there is insufficient blood flowto the brain to meet metabolic demand. This condition leads to pooroxygen supply or cerebral hypoxia and eventually the death of braintissue or ischemic stroke. There are two types of brain ischemia: (1)focal ischemia, which is confined to a specific region of the brain; and(2) global ischemia, which encompasses wide areas of brain tissue.

Peripheral artery disease (PAD) is a narrowing of the arteries otherthan those that supply the heart or the brain. Peripheral artery diseasemost commonly affects the legs and includes acute limb ischaemia,critical limb ischemia, renal ischemia, chronic mesenteric ischemia, andacute mesenteric ischemia.

As used herein, the term “effect angiogenesis” in a subject in needthereof refers to inducing a physiological process in which new bloodvessels form from pre-existing vessels. The formation of new bloodvessels may be indicated by: (1) an increase in a level of at least oneangiogenic protein (including several growth factors) such as fibroblastgrowth factor (FGF), vascular endothelial growth factor (VEGF), vascularendothelial growth factor receptor (VEGFR), neuropilin-1 (NRP-1),angiopoietin 1 (Ang1), angiopoietin 2 (Ang2), platelet-derived growthfactor (PDGF), platelet-derived growth factor receptor (PDGFR),transforming growth factor beta (TGF-β), transforming growth factor betareceptor, endoglin, chemokine ligand 2 (CCL2), integrins α_(v)β₃,α_(v)β₅ and α₅β₁, vascular endothelial cadherin, platelet endothelialcell adhesion molecule (PECAM-1), ephrin, plasminogen activators,plasminogen activator inhibitor-1, nitric oxide synthase 3 (eNOS),cyclooxygenase-2 (COX-2), AC133, DNA-binding protein inhibitor ID-1,DNA-binding protein inhibitor ID-3, and class 3 semaphorins. Theincrease in the level of the angiogenic protein may be at least 30%,40%, 50%, 60%, 70%, or 80%, relative to an initial angiogenic proteinlevel or an angiogenic protein level of a healthy subject (i.e., one whois not treated with Nigella sativa). In some embodiments, the increasein the angiogenic protein level may be accompanied by a reduction in avon Willebrand factor (VWF) level (by at least 30%, 40%, 50%, 60%, 70%,or 80%) relative to an initial VWF level or a VWF level in a healthysubject.

As used herein, the terms “treat”, “treatment”, and “treating” in thecontext of the administration of a therapy to a subject in need thereofrefer to the reduction or inhibition of the progression and/or durationof a disease, the reduction or amelioration of the severity of thedisease, and/or the amelioration of one or more symptoms thereofresulting from the administration of one or more therapies. “Treating”or “treatment” of the disease includes preventing the disease fromoccurring in a subject that may be predisposed to the disease but doesnot yet experience or exhibit symptoms of the disease (prophylactictreatment), inhibiting the disease (slowing or arresting itsdevelopment), ameliorating the disease, providing relief from thesymptoms or side-effects of the disease (including palliativetreatment), and relieving the disease (causing regression of thedisease). With regard to the disease, these terms simply mean that oneor more of the symptoms of the disease will be reduced. Such terms mayrefer to one, two, three, or more results following the administrationof one, two, three, or more therapies: (1) a stabilization, reduction(e.g., by more than 10%, 20%, 30%, 40%, 50%, preferably by more than 60%of the plaque in the arteries before administration), or elimination ofplaque; (2) inhibiting plaque buildup in arteries; (3) relieving to someextent (or, preferably, eliminating) one or more symptoms associatedwith a pathology related to or caused in part by plaque buildup inarteries; (4) an increase in disease-free, relapse-free,progression-free, and/or overall survival, duration, or rate; (5) adecrease in hospitalization rate; (6) a decrease in hospitalizationlength; (7) a stabilization or reduction in the rate of plaque buildup(e.g. by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, preferably at least80%) relative to the initial rate of plaque buildup in the arteries; (8)a reduction in the amount of calcium in the arteries (e.g. by at least10%, 20%, 30%, 40%, 50%, 60%, 70%, preferably by at least 80%) relativeto the initial amount of calcium; (9) a reduction in the totalcholesterol level in the blood (e.g. by at least 10%, 20%, 30%, 40%,50%, 60%, 70%, preferably by at least 80%) relative to the initial totalcholesterol level; (10) an impairment in the formation of plaque in thearteries; (11) a reduction in mortality; (12) a decrease in the need forsurgery; and (13) increase in blood supply to the heart, brain, legs,kidney, and/or intestine (by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,preferably by at least 80%) relative to the initial blood supply.

In the context of the disclosure, the term “arteries” refers to arteriesof the heart, brain (e.g., anterior cerebral artery, middle cerebralartery, and posterior cerebral artery), leg (e.g., femoral artery),kidney, and/or small intestine.

The results may be observed with methods such as a physical exam, bloodtest, electrocardiogram, chest X-ray, ankle/brachial index,echocardiography, computed tomography scan, stress testing, angiography,electromagnetic flowmeter, Doppler ultrasound, magnetic resonanceimaging (MRI), and positron emission tomography (PET). For example, theincrease in blood supply may be due to angiogenesis which may bedetermined by single photon emission computed tomography (SPECT) imagingand/or thrombolysis in myocardial infarction (TIMI) myocardial perfusiongrade. In other embodiments, the increase in blood supply may be due tothe reduction/removal of plaque from the arteries.

The terms “patient”, “subject”, and “individual” are usedinterchangeably. As used herein, they refer to individuals sufferingfrom a disease and encompass mammals and non-mammals. None of the termsrequire that the individual be under the care and/or supervision of amedical professional. Mammals are any member of the mammalian class,including but are not limited to humans, non-human primates, such aschimpanzees, and other apes and monkey species, farm animals, such ascattle, horses, sheep, goats, swine, domestic animals, such as rabbits,dogs, and cats, laboratory animals including rodents, such as rats, miceand guinea pigs, and the like. Examples of non-mammals include, but arenot limited to, birds, fishes, frogs, and the like. In preferredembodiments, the subject is a human. In some embodiments, the subject isa laboratory rat such as a biobreeding rat, a Brattleboro rat, ahairless rat, a knockout rat, a Lewis rat, a Long-Evans rat, a RoyalCollege of Surgeons (RCS) rat, a shaking rat Kawasaki, a Sprague Dawleyrat, a Wistar rat, or a Zucker rat. The rat may be male or female. Theage of the rat may be between 1-5 months, 1.5-4 months, or 2-3 months.In some embodiments, a weight of the rat falls within a healthy range of100-400 g, 150-350 g, or 200-300 g. In some embodiments, the rat isoverweight and weighs more than 400 g to 1.2 kg, 500-950 g, or 700-800g. In preferred embodiments, the laboratory rat is a Wistar rat.

A subject in need of treatment includes a subject already with thedisease, a subject which does not yet experience or exhibit symptoms ofthe disease, and a subject predisposed to the disease. In preferredembodiments, the subject is a person who is predisposed to ischemia,e.g. a person with high cholesterol levels and/or high blood pressure,sleep apnea, diabetes, and high blood triglyceride levels; a person whois overweight/obese, smokes, and/or has a sedentary lifestyle; or aperson with a family history of high cholesterol and/or high bloodpressure.

In another embodiment, the subject refers to a patient who has undergonesurgery (e.g., percutaneous coronary intervention (also known ascoronary angioplasty), artery bypass, stenting, and carotidendarterectomy) to treat atherosclerosis and/or ischemia.

Nigella sativa (also known as black seed) is a herb used as atraditional remedy in the Arab countries, Iran, Tunisia, and India. Itreceived its name from the appearance of the seeds which are small (1-5mg in weight) and have dark grey to black color (Kokoska L, Havlik J,Valterova I, Sovova H, Sajfrtova M, Jankovska I. Comparison of chemicalcomposition and antibacterial activity of Nigella sativa seed essentialoils obtained by different extraction methods. J Food Prot. 2008December; 71(12):2475-80. PubMed PMID: 19244901, incorporated herein byreference in its entirety). Generically, it is known as Nigella sativaLinn.

Broadly, Nigella sativa seeds contain 28-40%, 32-38%, or 36-38% fixedoil, 20-28% proteins, alkaloids (e.g., isoquinolinealkaloid—nigellicimine and nigellicimine n-oxide; pyrazolalkaloid—nigellidine and nigellicine), saponin, and 0.1-3%, 0.2-2.8%, or0.4-2.5% essential oil, based on a total weight of the seeds. Thecomposition of Nigella sativa grown in different parts of the world islisted in Table 1.

TABLE 1 Comparison of the composition of Nigella sativa obtained fromSaudi Arabia, Iran, and Tunisia Saudi Arabia Iran Tunisia Protein 20.85%22.6%, 26.7% Fixed and essential oil 38.20% 40.35% 28.48% Ash 4.37%4.41% 4.86% Total carbohydrate 31.94% 32.7% 40.0%

The fixed oil contains mainly of unsaturated fatty acids, includingarachidonic acid, eicosadienoic acid, linoleic acid, linolenic acid,oleic acid, palmitoleic acid, palmitic acid, stearic acid, and myristicacid. The fixed oil also contains beta-sitosterol, cyclocucalenol,cycloartenol, sterol esters, and sterol glucosides. The composition ofthe fixed oil from Nigella sativa grown in different parts of the worldis listed in Table 2.

TABLE 2 Comparison of the composition of fixed oil Nigella sativaobtained from Saudi Arabia, Iran, and Tunisia Saudi Arabia Tunisia Iranlinoleic acid 59.34% 50.3% 49.2% oleic acid 23.58% 25.0% 23.7% palmiticacid 11.9% 17.2% 18.4%

The following results (on a dry-weight basis) were obtained for Tunisianand Iranian Nigella sativa, respectively: protein 26.7% and 22.6%, oil28.48% and 40.35%, ash 4.86% and 4.41%, and total carbohydrates 40.0%and 32.7%. The major unsaturated fatty acids were linoleic acid (50.3%,49.2%), followed by oleic acid (25.0%, 23.7%), while the main saturatedfatty acid was palmitic acid (17.2%, 18.4%) for Nigella sativa grown inTunisia and Iran, respectively. Myristic, myristoleic, palmitoleic,margaric, margaroleic, stearic, linolenic, arachidic, eicosenoic,behenic and lignoceric acids were also detected (Cheikh-Rouhou S, BesbesS, Hentati B, Blecker C, Deroanne C, Attia H. Nigella sativa L.:Chemical composition and physicochemical characteristics of lipidfraction. Food Chemistry 2007; 101(2):673-681, incorporated herein byreference in its entirety).

In preferred embodiments, Nigella sativa grown in Saudi Arabia are usedin the method disclosed herein. Proximate analysis of black cumin seedsshowed a composition of 20.85 wt % protein, 38.20 wt % fat, 4.64 wt %moisture, 4.37 wt % ash, 7.94 wt % crude fibre and 31.94 wt % totalcarbohydrates, based on a total weight of Nigella sativa seeds(Al-Jassir M. Chemical composition and microflora of black cumin(Nigella sativa L.) seeds growing in Saudi Arabia. Food chemistry. 1992;45(4):239-42, incorporated herein by reference in its entirety).Potassium, phosphorus, sodium and iron were the predominant elementspresent. Zinc, calcium, magnesium, manganese and copper were found atlower levels. However, lead, cadmium and arsenic were not detected inthe seeds. Linoleic and oleic acids were the major unsaturated fattyacids while palmitic acid was the main saturated one. Glutamic acid,arginine and aspartic acid were the main amino acids present whilecystine and methionine were the minor amino acids. These resultsindicate the high nutritional potential of Saudi black cumin seedsespecially as a source of protein and fat. Thus, Nigella sativa fromSaudi Arabia may be preferred over other sources because the plantcontains high amounts of linoleic acid. For example, an amount oflinoleic acid in the fixed oil derived from Nigella sativa from SaudiArabia may be 5-30 percentage points, 8-20 percentage points, or 10-15percentage points higher than corresponding amounts in the fixed oilderived from Nigella sativa from other parts of the world.

The essential oil of Nigella sativa includes thymoquinone (10-70%,20-60%, or 27-57%), p-cymene (1-20%, 5-17%, or 7.1-15.5%), carvacrol(1-20%, 3-14%, or 5.8-11.6%), trans-anethole (0.1-5%, 0.2-4%, or0.25-2.3%), p-terpineol (1-10%, 1.5-8%, or 2-6.6%), longifoline(0.5-10%, or 1-8%), each based on a total volume of the essential oil.The essential oil also contains nigellone, thymohydroquinone, thymol,α-pinene, β-pinene, d-limonene, d-citronellol, and 4-terpineol.Thymoquinone readily dimerizes to form dithymoquinone and in the contextof the present disclosure, thymoquinone also refers to the dimerdithymoquinone.

Nigella sativa and its active component (thymoquinone) demonstratedmultiple therapeutic effects in several studies. Nigella sativa isanti-inflammatory, antioxidant, antibacterial, hypoglycemic, andantilipidemic (Hajhashemi V, Ghannadi A, Jafarabadi H. Black cumin seedessential oil, as a potent analgesic and anti-inflammatory drug.Phytother Res. 2004 March, 18(3):195-9. PubMed PMID: 15103664; Mansour MA, Nagi M N, El-Khatib A S, Al-Bekairi A M. Effects of thymoquinone onantioxidant enzyme activities, lipid peroxidation and DT-diaphorase indifferent tissues of mice: a possible mechanism of action. Cell BiochemFunct. 2002 June; 20(2):143-51. PubMed PMID: 11979510; Hawsawi Z A, AliB A, Bamosa A O. Effect of Nigella sativa (black seed) and thymoquinoneon blood glucose in albino rats. Ann Saudi Med. 2001 2001 May-July;21(3-4):242-4. PubMed PMID: 17264566; and Ali B H, Blunden G.Pharmacological and toxicological properties of Nigella sativa.Phytother Res. 2003 April; 17(4):299-305. PubMed PMID: 12722128, eachincorporated herein by reference in their entirety). Thymoquinone wasisolated and recognized as an important and active constituent of boththe essential and fixed oils (Salma Cheikh-Rouhoua, Souhail Besbesa,Basma Hentatib, Christophe Bleckerc, Claude Deroannec, Hamadi Attiaa.Nigella sativa L: Chemical composition and physicochemicalcharacteristics of lipid fraction. 2007. p. 673-81, incorporated hereinby reference in its entirety).

Nigella sativa was found to have antihypertensive effects. Two previousstudies reported antihypertensive effect on Nigella sativa on human andanimals (Dehkordi F R, Kamkhah A F. Antihypertensive effect of Nigellasativa seed extract in patients with mild hypertension. Fundam ClinPharmacol. 2008 August; 22(4):447-52. PubMed PMID: 18705755; and KhattabM M, Nagi M N. Thymoquinone supplementation attenuates hypertension andrenal damage in nitric oxide deficient hypertensive rats. Phytother Res.2007 May; 21(5):410-4. PubMed PMID: 17236176, each incorporated hereinby reference in their entirety). Cardiac adaptation for long termadministration of Nigella sativa was also demonstrated. In a studyperformed by El Bahai et al. long-term administration of Nigella sativafor two months for adult Wistar rats, lead to increases in the peakforce of contraction, myocardial flow rate and the rate of tensiondevelopment (El-Bahai M N, Al-Hariri M T, Yar T, Bamosa A O. Cardiacinotropic and hypertrophic effects of Nigella sativa supplementation inrats. Int J Cardiol. 2009 January; 131(3):e115-7. PubMed PMID: 17931720,incorporated herein by reference in its entirety). Another studyperformed by the same group, showed structural remodeling of themyocardium, including myocardial hypertrophy manifested by increasedheart weight/body weight (HW/BW), left ventricular weight/body weight(LVW/BW), and increased diameters of cardiomyocytes when Nigella sativawas fed to rats which were exercise-trained (Al-Asoom L I, Al-Shaikh BA, Bamosa A O, El-Bahai M N. Effect of Nigella sativa supplementation toexercise training in a novel model of physiological cardiac hypertrophy.Cardiovasc Toxicol. 2014 September; 14(3):243-50. PubMed PMID: 24497112,incorporated herein by reference in its entirety). The histological andelectrophysiological characteristics of hypertrophied hearts induced byNigella sativa in combination with exercise were compared toexercise-induced cardiac hypertrophy, and multiple similarities werefound (Al-Asoom L I, Al-Shaikh B A, Bamosa A O, El-Bahai M N. Comparisonof Nigella sativa- and exercise-induced models of cardiac hypertrophy:structural and electrophysiological features. Cardiovasc Toxicol. 2014September; 14(3):208-13. PubMed PMID: 24448711, incorporated herein byreference in its entirety). Furthermore, Ansam et al. reported aprotective effect of Nigella sativa against ischemia reperfusion insultin rat hearts when the animals fed Nigella sativa for three months (SeifA A. Nigella sativa attenuates myocardial ischemic reperfusion injury inrats. J Physiol Biochem. 2013 December; 69(4):937-44. PubMed PMID:23846789, incorporated herein by reference in its entirety).

However, none of the literature reported coronary angiogenesis occurringas a result of administering Nigella sativa or that Nigella sativa maybe useful for treating ischemic heart disease. In addition, the methoddisclosed herein effects coronary angiogenesis without causing cardiachypertrophy, which is unexpected. In the present disclosure, theangiogenic and therapeutic effects of Nigella sativa may be at leastpartially attributed to thymoquinone.

An essential oil, a plant extract, or a part of Nigella sativa plant maybe administered to the subject. Essential oils are aromatic oily liquidsobtained from plant materials (e.g., the whole plant, flowers, buds,seeds, leaves, twigs, stem, bark, fruits, roots, and combinationsthereof). Preferably, the essential oil is extracted from Nigella sativaseeds. Essential oils are a rich source of biologically active compounds(e.g., hydrocarbons, such as monoterpenes and sesquiterpenes; andoxygenated compounds, such as monoterpenoids, sesquiterpenoids, esters,ethers, aldehydes, ketones, and oxides).

Plant extracts, as defined herein, are not “essential oils” as notedabove. The plant extract may be obtained from any of the plant partsincluding the leaves, pulp, seeds, stems, fruit and fruit seeds, as wellas the whole plant. The plant extract comprises phytochemicals and/ormetabolites that include, without limitation, alkaloids, flavonoids,saponins, carbohydrates, polysaccharides, terpenoids (e.g.,monoterpenoids and sesquiterpenoids), steroids, sterols, phenols,tannins, anthraquinones, anthocyanins, amino acids, proteins, andvitamins.

In some embodiments, a part of the Nigella sativa plant (e.g., flowers,buds, seeds, leaves, twigs, stem, bark, fruits, roots, and combinationsthereof) is administered to the subject. The plant part(s) of interestmay be collected and then washed thoroughly, preferably twice/thricewith water, to remove both epiphytes and necrotic plants; preferablyfollowed by washing with water to remove associated debris if any. Insome embodiments, tap water, distilled water, doubly distilled water,deionized water, deionized distilled water, or combinations thereof maybe used to wash the plant part(s) of interest. The water may be sterile.In one embodiment, the water may have a conductivity of less than 10μS·cm⁻¹, less than 5 μS·cm⁻¹, or less than 1 μS·cm⁻¹ at 20-30° C.;and/or a resistivity greater than 0.1 MΩ·cm, greater than 1 MΩ·cm,greater than 5 MΩ·cm, or greater than 10 MΩ·cm at 20-30° C.; and/or atotal solid concentration less than 5 mg/kg, less than 1 mg/kg, or lessthan 0.5 mg/kg; and/or a total organic carbon concentration less than1000 μg/L, less than 200 μg/L, or less than 50 μg/L. The clean and freshplant parts may be sun-dried or dried in the shade for 5-25 days, orpreferably 7-20 days, or more preferably 10-15 days, and then finelycut/chopped, or preferably ground/powdered/pulverized using, forexample, a domestic blender. In preferred embodiments, the clean Nigellasativa seeds may be ground with a flax mill, a spice mill, a coffeegrinder, a food processor, a pepper mill, or a mortar and pestle. Atleast 80%, at least 85%, at least 90%, at least 95%, at least 99% of theground Nigella sativa seeds may pass through a mesh size larger than 20,larger than 100, or larger than 200.

The essential oil of Nigella sativa may be obtained by expression,fermentation, distillation, steam distillation, pressing, organicextraction, supercritical CO₂ extraction, or additional extractionmethods known to those familiar in the art. For example, the essentialoil may be obtained by extracting fresh/dried/ground plant part(s) ofinterest with supercritical CO₂ at a pressure of 140-300 bar, 180-250bar, or 200-220 bar, at 50-80° C., or 55-70° C., for 30-240 minutes,60-180 minutes, or 100-150 minutes.

The Nigella sativa plant may be at any growth stage, e.g. at a floweringstage when the flower and/or one or more non-flower parts (e.g. leaf,stem, and root) may be used to make the essential oil and/or the aqueousextract, or alternatively, at a non-flowering stage, when one or morenon-flower parts may be used to make the essential oil and/or theaqueous extract.

The aqueous extract may be prepared as follows. The fresh/dried/groundplant part(s) may be mixed with water (e.g., deionized distilled water)in an amount of 0.01-5 g/ml, 0.05-3 g/ml, 0.1-2 g/ml or 0.2-1 g/ml ofwater. The resulting mixture may be heated at a temperature of 60-100°C., 70-90° C., or 75-85° C. for 1-30 minutes, preferably 5-25 minutes,or more preferably 10-20 minutes. In some embodiments, the hot waterpercolation method is used. The aqueous extract may be administered asit is to the subject. In some embodiments, the resulting infusion isfiltered until no insoluble material appears in the aqueous extractprior to the administering.

In some embodiments, the ground plant part(s) or composition is freshlyprepared prior to every administering. The ground plant part(s) orcomposition may be administered 0.1-120 s, 1-80 s, or 10-60 s afterpreparation. In some embodiments, the ground plant part(s) orcomposition is administered 2-15 minutes, 5-12 minutes, or 8-10 minutesafter preparation.

In most embodiments, the Nigella sativa is formulated in a composition.As used herein, a “composition” refers to a mixture of the Nigellasativa with other chemical components, such as pharmaceuticallyacceptable carriers and excipients (either inert or active), making thecomposition especially suitable for therapeutic use in vivo. One purposeof a composition is to facilitate administration of the Nigella sativato the subject. The composition may contain 1-50 wt % Nigella sativa,3-40 wt %, 6-20 wt %, or 8-15 wt %, based on a total weight of thecomposition. In most embodiments, the composition consists essentiallyof Nigella sativa and water. Ingredients which do not affectangiogenesis may be included in such compositions.

As used herein, the phrase “pharmaceutically acceptable” refers to thecomposition is within the scope of sound medical judgment, suitable foruse in contact with the tissues of human beings and animals withoutexcessive toxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

As used herein, a “pharmaceutically acceptable carrier” refers to acarrier or diluent that does not cause significant irritation to anorganism, does not abrogate the biological activity and properties ofthe administered Nigella sativa, and/or does not interact in adeleterious manner with the other components of the composition in whichit is contained. The term “carrier” encompasses any excipient, binder,diluent, filler, salt (e.g., sodium chloride), buffer (e.g., phosphatebuffered saline), solubilizer, lipid, stabilizer, or other material wellknown in the art for use in pharmaceutical formulations. The choice of acarrier for use in a composition will depend upon the intended route ofadministration for the composition. The preparation of pharmaceuticallyacceptable carriers and formulations containing these materials isdescribed in, e.g. Remington's Pharmaceutical Sciences, 21st Edition,ed. University of the Sciences in Philadelphia, Lippincott, Williams &Wilkins, Philadelphia Pa., 2005, which is incorporated herein byreference in its entirety). Examples of physiologically acceptablecarriers include buffers such as phosphate buffers, citrate buffer, andbuffers with other organic acids; antioxidants including ascorbic acid;low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as TWEEN® (ICI, Inc.; Bridgewater, N.J.), polyethyleneglycol (PEG), and PLURONICS™ (BASF; Florham Park, N.J.). An “excipient”refers to an inert substance added to a composition to furtherfacilitate administration of Nigella sativa. Examples, withoutlimitation, of excipients include calcium carbonate, calcium phosphate,various sugars and types of starch, cellulose derivatives, gelatin,vegetable oils, and polyethylene glycols.

As used herein, the terms “therapies” and “therapy” can refer to anymethod or composition that can be used in the treatment and/ormanagement of the disease or one or more symptoms thereof. In someembodiments, the method disclosed herein may be administered to thesubject before and/or after surgeries such as angioplasty (e.g.,percutaneous coronary intervention, also known as coronary angioplasty),bypass grafting, and carotid endarterectomy. For example, the subjectmay be administered with Nigella sativa for up to 8 weeks, 6 weeks, or 2weeks before the surgery.

In some embodiments, the method disclosed herein is used in conjunctionwith prescribed medicine for high cholesterol and/or high blood pressure(e.g., statins, bile acid sequestrants, cholesterol absorptioninhibitors, fibrates, niacin, thiazide diuretics, omega-3 fatty acidsupplements, beta blockers, angiotensin-converting enzyme (ACE)inhibitors, angiotensin II receptor blockers (ARBs), calcium channelblockers, alpha blockers, alpha-beta blockers, central-acting agents,vasodilators, aldosterone antagonists, and renin inhibitors). Theadministration of the prescribed medicine may be concurrent with Nigellasativa or staggered.

In some embodiments, the method includes administering a second plant tothe subject. The amount of the second plant may range from 1-100 wt %,3-70 wt %, 6-50 wt %, or 10-30 wt % relative to the weight of Nigellasativa of the first effective amount. Preferably, the second plant alsocontains thymoquinone. Examples of second plants include, withoutlimitation, Monarda didyma, Monarda media, Monarda menthifolia, Thymuspulegioides, Thymus serpyllum, Thymus vulgaris, Satureja hortensis,Satureja montana, Eupatorium cannabinum, and Juniperus communis. Anessential oil, a plant extract, or a part (fresh/dried/ground) of thesecond plant may be administered. The administration of the second plantmay be concurrent with Nigella sativa or staggered.

A time interval between the administration of Nigella sativa and theadministration of the prescribed medicine and/or the second plant may beabout 1-5 minutes, 1-30 minutes, 30 minutes to 60 minutes, 1 hour, 1-2hours, or 2-6 hours.

The terms “administer”, “administering”, “administration”, and the like,as used herein, refer to the methods that may be used to enable deliveryof the Nigella sativa and/or the composition to the desired site ofbiological action. Routes or modes of administration are as set forthherein. These methods include, but are not limited to, oral routes orvia a gastrostomy tube. Those of ordinary skill in the art are familiarwith administration techniques that can be employed with the compositionand methods described herein. In preferred embodiments, the Nigellasativa and/or the composition described herein are administered orally.

Solid dosage forms for oral administration can include capsules,tablets, pills, powders, and granules. In such solid dosage forms,Nigella sativa is ordinarily combined with one or more adjuvantsappropriate to the indicated route of administration. Nigella sativa canbe admixed with lactose, sucrose, starch powder, cellulose esters ofalkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesiumstearate, magnesium oxide, sodium and calcium salts of phosphoric andsulfuric acids, gelatin, acacia gum, sodium alginate,polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted orencapsulated for convenient administration. Such capsules or tablets cancontain a controlled-release formulation as can be provided in adispersion of pulverized parts of Nigella sativa in hydroxypropylmethylcellulose. In the case of capsules, tablets, and pills, the dosage formscan also comprise buffering ingredients such as sodium citrate,magnesium or calcium carbonate or bicarbonate. Tablets and pills canadditionally be prepared with enteric coatings.

Liquid dosage forms for oral administration can include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions can also comprise adjuvants, such as wetting ingredients,emulsifying and suspending ingredients, and sweetening, flavoring, andperfuming ingredients.

Emulsifiers may be added for stability of the final product. Examples ofsuitable emulsifiers include, but are not limited to, lecithin (e.g.,from egg or soy), or mono- and di-glycerides. Other emulsifiers arereadily apparent to the skilled artisan and selection of suitableemulsifier(s) will depend, in part, upon the formulation and finalproduct.

In addition to the carbohydrates described herein, the composition cancontain natural or artificial sweeteners, e.g., sucrose, fructose,cyclamates, asparagine, sucralose, aspartame, and acesulfame K.

For therapeutic purposes, formulations for administering via thegastrostomy tube can be in the form of aqueous or non-aqueous isotonicsterile solutions or suspensions. These solutions and suspensions can beprepared from sterile Nigella sativa powder or granules and one or moreof the carriers or diluents mentioned for use in the formulations fororal administration. Nigella sativa can be mixed with water,polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseedoil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, and/orvarious buffers. Other adjuvants and modes of administration are welland widely known in the pharmaceutical art. The sterilesolution/suspension may be prepared in a non-toxic parenterallyacceptable diluent or solvent, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution, and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilcan be employed including synthetic mono- or diglycerides. In addition,fatty acids, such as oleic acid, find use in the preparation of sterilesolution/suspension. Dimethyl acetamide, surfactants including ionic andnon-ionic detergents, polyethylene glycols can be used. Mixtures ofsolvents and wetting ingredients such as those discussed above are alsouseful.

In other embodiments, Nigella sativa is provided as dietary supplementin capsule or tablet form. Nigella sativa can be formulated into a foodor drink, and provided, for example, as a snack bar, a cereal, a drink,or in any other easily ingested form. Flavors, coloring agents, spices,nuts and the like can be incorporated into the product. Flavorings canbe in the form of flavored extracts, essential oils, chocolateflavorings (e.g., non-caffeinated cocoa or chocolate, chocolatesubstitutes such as carob), peanut butter flavoring, cookie crumbs,crisp rice, vanilla or any commercially available flavoring. Flavoringscan be protected with mixed tocopherols. Examples of useful flavoringsinclude but are not limited to pure anise extract, imitation bananaextract, imitation cherry extract, chocolate extract, pure lemonextract, pure orange extract, pure peppermint extract, imitationpineapple extract, imitation rum extract, imitation strawberry extract,or pure vanilla extract; or essential oils, such as balm oil, bay oil,bergamot oil, cedar wood oil, cherry oil, walnut oil, cinnamon oil,clove oil, or peppermint oil; peanut butter, chocolate flavoring,vanilla cookie crumb, butterscotch or toffee. In a preferred embodiment,the dietary supplement contains berry or other fruit flavor. The foodcompositions may further be coated, for example with a yogurt coating ifit is as a bar.

Preservatives may also be added to the composition to extend productshelf life. Preferably, preservatives such as potassium sorbate, sodiumsorbate, potassium benzoate, and sodium benzoate are used.

As used herein, the term “beverage” means a consumer ready productcomprising any liquid intended for human or animal consumption.Exemplary types of beverages include, but are not limited to, berriesand small fruit juices, smoothies, citrus fruit juices, dairy products(i.e. milks, yogurts, and soy beverages), grape juices, syrups, wines,beers, teas, coffees, cocoas/chocolates, tomato juices, and the like.The beverage contain Nigella sativa and may contain other ingredientssuch as vitamins, natural and artificial flavors, electrolytes, dietaryfiber, calcium, iron, zinc, calcium disodium EDTA, citric acid, CO₂, thesecond plant, or any other desired ingredient.

In one embodiment, the beverage is a tea, an aromatic beverage commonlyprepared by pouring hot or boiling water over a plant or plant parts.For example, the fresh/dried/ground plant part(s) may be mixed withboiled water (the temperature of the water may be 80-100° C., 90-100°C., preferably 95-100° C.) and left to steep for 1-5 minutes, 2-4minutes, or about 3 minutes. In certain embodiments, the beverage of thepresent disclosure may further comprise one or more plants, includingherbal tea plants. Exemplary plants include, but are not limited to,anise (seeds or leaves), asiatic penny-wort leaf, artichoke, bee balm,boldo, burdock, caraway, catnip, chamomile, che dang (ilex causueleaves), chinese knot-weed, chrysanthemum, cinnamon, cocoa, coffeeleaves and coffee cherry, cerasse, citrus peel (including bergamot,lemon and orange peels), dandelion coffee, dill, echinacea, elderberry,european mistletoe (viscum album), essiac, fennel, gentian, ginger root,ginseng, goji, hawthorn, hibiscus, ho yan hor herbal, honeybush,horehound, houttuynia, hydrangea (hydrangea serrata amacha), jiaogulan,kapor, kava root, kratom, kuzuyu, labrador, lapacho (also known astaheebo), lemon balm, lemon and ginger, lemon grass, luo han guo,licorice root, lime blossom, mint, mountain, neem leaf, nettle leaf, newjersey, noni, oksusu cha, pennyroyal leaf, pine, qishr, red clover, redraspberry leaf, roasted barley, roasted wheat, rooibos (red bush), rosehip, roselle petals (species of hibiscus; aka bissap, dah, etc.),rosemary, sagebrush, california sagebrush, sage, sakurayu, salvia,scorched rice, skullcap, sobacha, spicebush (lindera benzoin), spruce,staghorn sumac fruit, stevia, St. John's wort, thyme, tulsi, holy basil,uncaria tomentosa (commonly known as cat's claw), valerian, verbena(vervains), vetiver, wax gourd, wong lo kat, woodruff, yarrow, and thelike and mixtures thereof.

The first aspect of the disclosure relates to a method for effectingangiogenesis and optionally treating ischemia. The method comprisesadministering a first effective amount of Nigella sativa to a subject inneed thereof.

The terms “effective amount”, “therapeutically effective amount”, or“pharmaceutically effective amount” refer to that amount of the Nigellasativa being administered which will relieve to some extent one or moreof the symptoms of the disease being treated. The result can bereduction and/or alleviation of the signs, symptoms, or causes of adisease, or any other desired alteration of a biological system. Anappropriate “effective amount” may differ from one individual toanother. An appropriate “effective amount” in any individual case may bedetermined using techniques, such as a dose escalation study. The dosageand treatment duration are dependent on factors, such as bioavailabilityof a drug, administration mode, toxicity of a drug, gender, age,lifestyle, body weight, the use of other drugs and dietary supplements,the disease stage, tolerance and resistance of the body to theadministered drug, etc., and then determined and adjusted accordingly.

The first effective amount may be in a range of 0.1-1 g/kg body weightof the subject, 0.3-0.95 g/kg, 0.6-0.9 g/kg, or 0.7-0.85 g/kg. The firsteffective amount of Nigella sativa may be administered 1-3 times a day,1-2 times/day, or preferably once a day. The first effective amount ofNigella sativa may be administered 2-6 days, 3-6 days, or 4-6 days perweek for a period of 4-52 weeks, 5-40 weeks, 6-30 weeks, or preferably7-10 weeks. In preferred embodiments, the first effective amount ofNigella sativa is administered for 4-6 consecutive days per week.Accordingly, a time lapse of 1-3 days is present between eachadministering. In some embodiments, the first effective amount isadministered once a week and may still effect coronary angiogenesis andoptionally treat ischemic heart disease.

In some preferred embodiments, this cycling method effects coronaryangiogenesis but does not lead to cardiac hypertrophy in the subject. Asused herein, the term “cardiac hypertrophy” refers to thickening of theheart muscle which results in a decrease in size of the chamber of theheart, including the left and right ventricles. Cardiac hypertrophy maybe determined with electrocardiography, ultrasound, or computedtomography. In some embodiments, cardiac hypertrophy is determined fromthe levels of PECAM-1 which is described hereinafter. Alternatively, thepresence of cardiac hypertrophy may be determined from the weight of theheart, the weight of each of the left and right ventricles, the ratiobetween the heart weight and the subject's weight, the ratio between theweight of the left/right ventricle and the subject's weight, and anaverage diameter of cardiomyocytes. An average diameter ofcardiomyocytes in a hypertrophic (i.e., enlarged) heart may be at least10%, 20%, 40%, 60%, or 80% larger than an average diameter ofcardiomyocytes in a normal heart. Normal cardiomyocytes may have anaverage diameter in a range of 10-25 μm, 12-22 μm, or 15-20 μm. As usedherein, the term “diameter” refers to the greatest possible distancemeasured from a first point on a cross-section of the cardiomyocytethrough the center of the cross-section to a second point directlyacross from the first point. An average weight of a normal adult humanheart may be 88-235 g, 100-200 g, or 160-180 g (R. M. Fulton, E. C.Hutchinson, And A. Morgan Jones, Ventricular Weight In CardiacHypertrophy, British Heart Journal, 1952, 14: 413-420, incorporatedherein by reference in its entirety). An average weight of a normalright ventricle may be 23-68 g, 30-60 g, or 40-50 g. An average weightof a normal left ventricle may be 48-123 g, 50-110 g, or 80-90 g. Anaverage weight of a heart with cardiac hypertrophy may weigh at least20%, at least 50%, or at least 80% heavier than a normal heart, forexample, 173-606 g, 200-500 g, or 220-400 g. An average weight of ahypertrophic right ventricle may weigh at least 20%, at least 50%, or atleast 70% heavier than a normal right ventricle, for example, 55-178 g,60-150 g, or 75-110 g. An average weight of a hypertrophic leftventricle may weigh at least 50%, at least 80%, or at least 90% heavierthan a normal left ventricle, for example, 121-346 g, 150-250 g, or170-220 g. In preferred embodiments, the weight of the subject's heartfalls within the weight range for a normal heart, or the weight of thesubject's left or right ventricle falls within the weight range for anormal left or right ventricle, respectively, before and after theadministration of Nigella sativa.

In most embodiments, the method further comprises measuring aconcentration of a biomarker and/or detecting a mutation in a biomarkerbefore and/or after the Nigella sativa is administered. As used herein,the term “biomarker” refers to a characteristic that is objectivelymeasured and evaluated as an indicator of normal biological processes,pathogenic processes or pharmacological responses to a therapeuticintervention. Exemplary biomarkers related to angiogenesis and/orischemia include, without limitation, angiogenic proteins (includingseveral growth factors) such as fibroblast growth factor (FGF), vascularendothelial growth factor (VEGF), vascular endothelial growth factorreceptor (VEGFR), neuropilin-1 (NRP-1), angiopoietin 1 (Ang1),angiopoietin 2 (Ang2), platelet-derived growth factor (PDGF),platelet-derived growth factor receptor (PDGFR), transforming growthfactor beta (TGF-β), transforming growth factor beta receptor, endoglin,chemokine ligand 2 (CCL2), integrins α_(v)β₃, α_(v)β₅ and α₅β₁, vascularendothelial cadherin, platelet endothelial cell adhesion molecule(PECAM-1), ephrin, plasminogen activators, plasminogen activatorinhibitor-1, nitric oxide synthase 3 (eNOS), cyclooxygenase-2 (COX-2),AC133, DNA-binding protein inhibitor ID-1, DNA-binding protein inhibitorID-3, and class 3 semaphorins; myeloperoxidase (MPO); cardiac troponin T(cTnT) and troponin I (cTnI); C-reactive protein (CRP); and otherinflammatory biomarkers such as cytokines (interleukin (IL)-1, IL-6,IL-8, monocyte chemoattractant protein-1 (MCP-1)), soluble CD40 ligand,serum amyloid A (SAA), selectins (E-selectin, P-selectin), matrixmetalloproteinases (MMPs), cellular adhesion molecules (intercellularadhesion molecule 1 (ICAM-1), and vascular adhesion molecule 1(VCAM-1)); placental growth factor (PIGF); A2 phospholipases;low-density lipoprotein (LDL) receptor gene; APOE gene;lecithin-cholesterol acyltransferase gene; and ATP binding cassettetransporter (ABCA1) gene.

VEGF is a growth factor that induces the sprouting of the capillaries.It does so by dissolving the basement membrane of the existingcapillaries, and then stimulating the proliferation and migration ofendothelial cells (Zheng W, Seftor E A, Meininger C J, Hendrix M J,Tomanek R J. Mechanisms of coronary angiogenesis in response to stretch:role of VEGF and TGF-beta. Am J Physiol Heart Circ Physiol. 2001February; 280(2):H909-17. PubMed PMID: 11158993, incorporated herein byreference in its entirety). VEGF interacts with the endothelial celladhesion molecules during the process of endothelial growth andproliferation (Kolmakova A, Rajesh M, Zang D, Pili R, Chatterjee S. VEGFrecruits lactosylceramide to induce endothelial cell adhesion moleculeexpression and angiogenesis in vitro and in vivo. Glycoconj J. 2009July; 26(5):547-58. PubMed PMID: 19219548, incorporated herein byreference in its entirety). In one study, rats that exercised for fourweeks were found to have upregulation of VEGF, accompaniedsimultaneously with capillary sprouting and capillary angiogenesis(Al-Asoom L I, Al-Shaikh B A, Bamosa A O, El-Bahai M N. Comparison ofNigella sativa and exercise-induced models of cardiac hypertrophy:structural and electrophysiological features. Cardiovasc Toxicol. 2014September; 14(3):208-13, incorporated herein by reference in itsentirety). Induction of VEGF production in an endothelial cell culturehas been found to promote cell division and cell proliferation (Peng YZ, Zheng K, Yang P, Wang Y, Li R J, Li L, et al. Shock wave treatmentenhances endothelial proliferation via autocrine vascular endothelialgrowth factor. Genet Mol Res. 2015; 14(4):19203-10. PubMed PMID:26782573, incorporated herein by reference in its entirety). Nurro etal. showed that the administration of VEGF genes into porcine myocardiuminduced angiogenesis and increased the microvascular bed about 1.8 fold(Nurro J, Halonen P J, Kuivanen A, Tarkia M, Saraste A, Honkonen K, etal. AdVEGF-B186 and AdVEGF-DANAC induce angiogenesis and increaseperfusion in porcine myocardium. Heart. 2016 November; 102(21):1716-20.PubMed PMID: 27173504, incorporated herein by reference in itsentirety).

The process of angiogenesis is also governed by antiangiogenic factors.VWF is a glycoprotein that is synthesised in the endothelial cells, andit is known for its essential haemostatic effect. It stimulates theadhesion and aggregation of platelets in injured tissue, and it acts asa carrier of coagulation factor VIII (Randi A M, Laffan M A, Starke R D.Von Willebrand factor, angiodysplasia and angiogenesis. Mediterr JHematol Infect Dis. 2013; 5(1):e2013060. PubMed PMID: 24106610. PubmedCentral PMCID: PMC3787682, incorporated herein by reference in itsentirety). VWF plays a role in the regulation of angiogenesis, limitsthe release of angiogenic factors, such as VEGF, and stabilises thecapillary bed (Starke R D, Ferraro F, Paschalaki K E, Dryden N H,McKinnon T A, Sutton R E, et al. Endothelial von Willebrand factorregulates angiogenesis. Blood. 2011 January; 117(3):1071-80. PubMedPMID: 21048155. Pubmed Central PMCID: PMC3035068, incorporated herein byreference in its entirety). A deficiency in VWF results in abnormalgrowth of vessels in angiodysplasia (Bauditz J, Lochs H. Angiogenesisand vascular malformations: antiangiogenic drugs for treatment ofgastrointestinal bleeding. World J Gastroenterol. 2007 December;13(45):5979-84. PubMed PMID: 18023086. Pubmed Central PMCID: PMC4250877,incorporated herein by reference in its entirety). Inhibition of thesynthesis and release of VWF by interfering with its genetic expressionin mice leads to enhanced angiogenesis, increased VEGF receptor-2dependent proliferation and migration of endothelial cells.

Nitric oxide (NO) is a multifunctional cellular signalling molecule,known for its vasodilation action. Recently, evidence has shown that NOplays a role as a mediator of angiogenesis. NO is released from theendothelial cells under the influence of VEGF. VEGF upregulates andactivates nitric oxide synthase (NOS) in endothelial cells (Cooke J P,Losordo D W. Nitric oxide and angiogenesis. Circulation. 2002 May;105(18):2133-5. PubMed PMID: 11994243, incorporated herein by referencein its entirety). Increased expression of NOS was demonstrated in thehind-limbs of mice after three weeks of wheel running (Schirmer S H,Millenaar D N, Werner C, Schuh L, Degen A, Bettink S I, et al. Exercisepromotes collateral artery growth mediated by monocytic nitric oxide.Arterioscler Thromb Vasc Biol. 2015 August; 35(8):1862-71. PubMed PMID:26088573, incorporated herein by reference in its entirety).Overexpression of both endothelial and inducible NOS was alsodemonstrated in the heart after training in both mice and humans.Ischemia might be a crucial trigger for the release and synthesis ofinducible NOS (iNOS, also known as NOS-2), and its upregulation afterexercise training depends on the degree of ischemia induced by thetraining intensity (Kleindienst A, Battault S, Belaidi E, Tanguy S,Rosselin M, Boulghobra D, et al. Exercise does not activate the β3adrenergic receptor-eNOS pathway, but reduces inducible NOS expressionto protect the heart of obese diabetic mice. Basic Res Cardiol. 2016July; 111(4):40. PubMed PMID: 27164904, incorporated herein by referencein its entirety). Nigella sativa is known to be an anti-inflammatory andantioxidant mediator and it might not culminate in overexpression ofNOS-2.

PECAM-1 is the platelet-endothelial adhesion molecule known as CD31(Feng D, Nagy J A, Pyne K, Dvorak H F, Dvorak A M. Ultrastructurallocalization of platelet endothelial cell adhesion molecule (PECAM-1,CD31) in vascular endothelium. J Histochem Cytochem. 2004 January;52(1):87-101. PubMed PMID: 14688220, incorporated herein by reference inits entirety). The interaction of PECAM-1 with growth factors isessential for angiogenesis (DeLisser H M, Christofidou-Solomidou M,Strieter R M, Burdick M D, Robinson C S, Wexler R S, et al. Involvementof endothelial PECAM-1/CD31 in angiogenesis. Am J Pathol. 1997September; 151(3):671-7. PubMed PMID: 9284815. Pubmed Central PMCID:PMC1857836, incorporated herein by reference in its entirety). Somestudies have used PECAM-1 to estimate capillary density. In a recentstudy, diabetic rats were exposed to testosterone treatment and exerciseto investigate the effect on these two interventions on neoangiogenesis(Nurro J, Halonen P J, Kuivanen A, Tarkia M, Saraste A, Honkonen K, etal. AdVEGF-B186 and AdVEGF-DANAC induce angiogenesis and increaseperfusion in porcine myocardium. Heart. 2016 November; 102(21):1716-20,incorporated herein by reference in its entirety). That studydemonstrated more PECAM-1 antibody staining after both treatmentmodalities were administered (Chodari L, Mohammadi M, Ghorbanzadeh V,Dariushnejad H, Mohaddes G. Testosterone and Voluntary Exercise PromoteAngiogenesis in Hearts of Rats with Diabetes by Enhancing Expression ofVEGF-A and SDF-1a. Can J Diabetes. 2016 July PubMed PMID: 27444229,incorporated herein by reference in its entirety). Cardiac hypertrophyis associated with an augmented blood vessel density (Marc Thiriet,Diseases of the Cardiac Pump, Springer, 2015). As such high PECAM-1levels may be indicative of cardiac hypertrophy.

Cardiac troponins may be elevated in acute coronary syndromes and areused to assess ischemic heart diseases.

Myeloperoxidase (MPO) is involved in the inflammatory process thatprecedes the onset of symptomatic coronary artery disease (Meuwese M C,Stroes E S, Hazen S L et al.: Serum myeloperoxidase levels areassociated with the future risk of coronary artery disease in apparentlyhealthy individuals: the EPIC-Norfolk Prospective Population Study. J.Am. Coll. Cardiol. 50, 159-165 (2007), incorporated herein by referencein its entirety). Elevated levels of myeloperoxidase may predict futurerisk of coronary artery disease in apparently healthy individuals who donot show the symptoms.

The mutation in the biomarker may be detected with a polymerase chainreaction (PCR) assay, DNA microarray, multiplex ligation-dependent probeamplification (MLPA), single strand conformational polymorphism (SSCP),denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis,and restriction fragment length polymorphism (RFLP). The procedures todetect the mutation are well-known to those of ordinary skill in theart.

The concentration of the biomarker may be measured with an assay, forexample an antibody-based method (e.g., an ELISA). As used herein, theterm “antibody-based method” refers to any method with the use of anantibody including, but not limited to, enzyme-linked immunosorbentassay (ELISA), Western blotting, immunoprecipitation (IP), enzyme linkedimmunospot (ELISPOT), immunostaining, immunohistology(immunohistochemistry), immunocytochemistry, affinity chromatography,and the like.

In preferred embodiments, immunohistology is used to detect and quantifythe biomarkers in a sample. The biomarker may be detected with a director indirect method. The direct method is a one-step staining method andinvolves a labelled antibody (e.g. FITC-conjugated antibody) reactingdirectly with the biomarker in the sample. The indirect method involvesan unlabelled primary antibody that binds to the biomarker in the sampleand a labelled secondary antibody that reacts with the primary antibody.In some embodiments, after the biomarker has reacted with the antibody,a dye is applied to provide additional contrast. Exemplary dyes include,without limitation, hematoxylin, Hoechst stain, and DAPI.

The stained areas may be visualized by a confocal microscope or anoptical microscope. Soft copies of the micrographs may be obtained withthe microscopes or by digitizing images from hard copies of micrographs.A software, such as ImageJ or a proprietary software provided by themicroscope vendor, may be used to analyze/quantify the stained areas.The stained areas in each micrograph are expressed as a percentage ofthe total area of each micrograph. This percentage is proportional tothe amount of the biomarkers present in the sample.

In accordance with the method of quantification described in thepreceding paragraph, an amount of VEGF before administering the firsteffective amount or in a subject who is not treated with Nigella sativamay be in a range of 0.8-2.4%, 1-2%, or 1.5-1.6%. An amount of VEGFafter administering the first effective amount may be in a range of1.2-4%, 1.5-3.5%, 2-3%, or 2.5-2.7%.

An amount of VWF before administering the first effective amount or in asubject who is not treated with Nigella sativa may be in a range of0.6-4.1%, 2-3%, or 2.3-2.4%. An amount of VWF after administering thefirst effective amount may be in a range of 0.7-2.4%, 0.9-2%, 1.2-1.8%,or 1.4-1.6%.

An amount of PECAM-1 before administering the first effective amount orin a subject who is not treated with Nigella sativa may be in a range of0.02-2.36%, 0.5-2%, 1-1.5%, or 1.1-1.3%. In embodiments where the methodeffects coronary angiogenesis without causing cardiac hypertrophy, theamount of PECAM-1 is substantially the same (e.g., deviates less than15%, 10%, 5%, 3%, 2%, or 1%) as the amount of PECAM-1 beforeadministering the first effective amount. Elevated levels of PECAM-1 maybe in a range of 1-2.6%, 1.3-2%, or 1.6-1.9. In some embodiments wherethe sample contains heart cells, elevated levels of PECAM-1 indicatecardiac hypertrophy.

In preferred embodiments, an amount of NOS-2 before and afteradministering the first effective amount may be in a range of0.06-2.44%, 0.5-2%, 1-1.5%, or 1.1-1.3%.

In some embodiments, an ELISA is used. The term “ELISA” refers to amethod of detecting the presence and quantify of a biomarker in asample. There are several variants of ELISA, including, but not limitedto, sandwich ELISA, competitive ELISA, indirect ELISA, ELISA reverse,and the like. The ELISA assay may be a singleplex assay or a multiplexassay, which refers to a type of assay that simultaneously measuresmultiple analytes in a single run/cycle of the assay. Preferably, asandwich ELISA is used.

The protocol for measuring the concentration of the biomarker and/ordetecting the mutation in the biomarker is known to those of ordinaryskill, for example by performing the steps outlined in the commerciallyavailable assay kit sold by Sigma-Aldrich, Thermo Fisher Scientific, R &D Systems, ZeptoMetrix Inc., Cayman Inc., Abcam, Trevigen, DojindoMolecular Technologies, Biovision, and Enzo Life Sciences.

The term “sample” includes any biological sample taken from the subjectincluding cells (e.g., cardiac muscle cells, brain cells, kidney cells,and intestine cells), a tissue sample, or body fluid. For example, asample may include a skin sample, a cheek cell sample, saliva, or bloodcells. A sample can include, without limitation, a single cell, multiplecells, fragments of cells, an aliquot of a body fluid, whole blood,platelets, serum, plasma, red blood cells, white blood cells,endothelial cells, synovial fluid, and lymphatic fluid.

In some embodiments, the mutation in the biomarker is detected beforeadministrating the composition to identify subjects predisposed to thedisease. For example, subjects with a mutation in at least one of thegenes described herein are at a higher risk of contractingatherosclerosis which may lead to ischemia.

In some embodiments, the biomarkers are measured/detected after theadministration. For example, the measurement may be 1-5 minutes, 1-30minutes, 30-60 minutes, 1-2 hours, 2-12 hours, 12-24 hours, 1-2 days,3-6 days, 1-20 weeks, or any period of time in between after theadministering. In preferred embodiments, the biomarkers aremeasured/detected after the first effective amount of Nigella sativa hasbeen administered for 1-10 weeks, 2-9 weeks, or 6-8 weeks.

In some embodiments, the method further comprises administering a secondeffective amount of Nigella sativa after the concentration of thebiomarker is measured.

The second effective amount is different from the first effectiveamount. The second effective amount may be in a range of 0.1-1 g/kg bodyweight of the subject, 0.3-0.95 g/kg, 0.6-0.9 g/kg, or 0.7-0.85 g/kg.The second effective amount of Nigella sativa may be administered 1-3times a day, 1-2 times/day, or preferably once a day. The secondeffective amount may be administered 1-4 times a week, or 2-3 times aweek.

In embodiments where first and second effective amounts of Nigellasativa are administered, the first effective amount of Nigella sativaand/or the second effective amount of Nigella sativa is administered for1 day or 2-10, 3-7, or 4-6 consecutive days. The duration for which thesecond effective amount is administered may be the same or different asthe duration for the administration of the first effective amount.

The second effective amount, the administering frequency, and/or theadministering duration may be adjusted according to the concentration ofthe biomarkers measured before and after the administration of the firsteffective amount. When the concentration of the biomarkers associatedwith angiogenesis and/or ischemia does not increase/decrease by 1-40%,15-30%, or 20-25%, relative to the initial concentration of thebiomarkers, the second effective amount is increased by 5-200%, 30-150%,50-100% relative to the first effective amount, the administeringfrequency is 2-5 times, or 3-4 times more frequent than theadministering frequency of the first effective amount, and/or the secondeffective amount is administered daily. In these embodiments, the secondeffective amount may range from 0.1-2 g/kg body weight, 0.3-1.9 g/kg,0.6-1.8 g/kg, or 0.7-1.7 g/kg. When the concentration of the biomarkersassociated with angiogenesis and/or ischemia increases/decreases by morethan 40%, or 45-70%, or 50-60%, relative to the initial concentration ofbiomarkers, the second effective amount is reduced to 5-90%, 10-60%, or15-30% relative to the first effective amount. In these embodiments, thesecond effective amount may range from 0.01-0.95 g/kg body weight,0.04-0.9 g/kg, 0.07-0.85 g/kg. Also, there may be 2-5, or 3-4 fewerdosages per week of the second effective amount compared to the numberof weekly dosage of the first effective amount.

In some embodiments, the administration of Nigella sativa is stoppedonce angiogenesis is effected and/or the subject is treated.

Having generally described this disclosure, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

Example 1 Animals

Fifteen normal adult male Wistar rats (age: two months, weight: 200-300g) were obtained from the animal house at the University of Dammam, inDammam, Saudi Arabia. All the animals were housed individually inlabelled cages with adequate ventilation and illumination, and in normallight cycle (12 light/12 dark). Normal laboratory chow and tap waterwere available ad libitum. The sample size was selected based on apublished work with the consideration of the minimum requirement toachieve meaningful results (Chen H I, Sharma B, Akerberg B N, Numi H J,Kivelä R, Saharinen P, et al. The sinus venosus contributes to coronaryvasculature through VEGFC-stimulated angiogenesis. Development. 2014December; 141(23):4500-12. PubMed PMID: 25377552. Pubmed Central PMCID:PMC4302936, incorporated herein by reference in its entirety).

Ethical approval was provided by the ethical committee of the Deanshipof Scientific Research at Dammam University with reference numberIRB-2014-01-165. This committee is a branch of the National Committee ofBioethics, Saudi Arabia.

The rats were divided equally and randomly into three groups: thecontrol group, the Nigella sativa (NS)-treated group, and theexercise-trained group. The NS-treated rats were given 800 mg/kg Nigellasativa on every weekday for eight weeks. Nigella sativa whole seeds wereobtained from a local herb store in Dammam, Saudi Arabia. It is aproduct of the central region of Saudi Arabia. These seeds werepreviously analysed for its constituents by Al-Jassir (Al-Jassir M.Chemical composition and microflora of black cumin (Nigella sativa L.)seeds growing in Saudi Arabia. Food chemistry. 1992; 45(4):239-42,incorporated herein by reference in its entirety). The dose, durationand preparation of Nigella sativa was similar to that used by El Bahaiet al., who showed cardiac hypertrophy and positive inotropic effect inthe subjects after treatment. Oral administration was done via a feedingneedle. A suspension of Nigella sativa was prepared by dissolving 10 gof freshly ground Nigella sativa seeds in 100 ml of distilled water. Theanimals in the control group were given an equivalent volume of water.

The rats in the exercise group were trained on a treadmill (IITC LifeScience, five-lane rat treadmill), five days/week for eight weeks. Thespeed, grade and duration were progressively increased during the firstweek until the final protocol was achieved, with a speed of 18 m/min andan inclination of 30-32°, for a two-hour session (Barbier J,Rannou-Bekono F, Marchais J, Tanguy S, Carré F. Alterations ofbeta3-adrenoceptors expression and their myocardial functional effectsin physiological model of chronic exercise-induced cardiac hypertrophy.Mol Cell Biochem. 2007 June; 300(1-2):69-75. PubMed PMID: 17131040,incorporated herein by reference in its entirety). An electric grid atthe rear of the belt was used as the running stimulus. An equivalentvolume of water was also administered to this group.

All rat groups were matched in regard to the initial and the final bodyweight.

Example 2 Extraction of the Hearts

Surgical extraction of the hearts was done using a longitudinalabdominal incision. Upon excision, the heart was soaked in cold Ringer'ssolution. Excess connective tissue and great vessels were removed.Lumens were rinsed with Ringer's solution to remove excess blood andclots. Finally, the hearts were blotted dry and weighed. The rightventricles and both atria were removed, and the remaining leftventricles were weighed. The free wall of the left ventricles wasexcised and stored in 4% formal-saline for histological preparation.

Example 3 Preparation of the Light Microscope Slides

The left ventricles were washed briefly in water, and then labelled andplaced in a tissue processor (Tissue-Tek VIP) overnight. The specimenswere dehydrated using ascending grades of alcohol: 70%, 90%, and 100%(two changes for 100% alcohol), and two changes of xylene for a periodof two hours each.

The process of embedding the specimens was initiated. The specimens wereimpregnated in two changes of molten paraffin wax for a period of twohours for each change, and they were subsequently embedded in moltenparaffin wax at a temperature of 60° C. Cassettes were used to controlthe position of the specimens. The wax blocks of tissues were labelled,allowed to solidify and then sectioned using microtomy (LEICA RM 2235,Leica BioSystems Buffalo Grove, Ill., USA) at a thickness of 3 μm. Thesectioned tissues were floated in warm water, and then placed onmicroscope slides, labelled and allowed to dry.

The sections were dewaxed, washed in water and stained using thefollowing antibodies:

a. Rat VEGF 164 affinity purified polyclonal antibody (Ab), Goat IgG(R&D Systems, Minneapolis, Minn., USA) (Lu J, Yao Y Y, Dai Q M, Ma G S,Zhang S F, Cao L, et al. Erythropoietin attenuates cardiac dysfunctionby increasing myocardial angiogenesis and inhibiting interstitialfibrosis in diabetic rats. Cardiovasc Diabetol. 2012; 11:105. PubMedPMID: 22954171. Pubmed Central PMCID: PMC3527329, incorporated herein byreference in its entirety).

b. Anti-CD31 (PECAM-1) for endothelial cells to determine the density ofthe capillaries, purified polyclonal Ab, Goat IgG (R&D Systems,Minneapolis, Minn., USA) (Ainscough J F, Drinkhill M J, Sedo A, Turner NA, Brooke D A, Balmforth A J, et al. Angiotensin II type-1 receptoractivation in the adult heart causes blood pressure-independenthypertrophy and cardiac dysfunction. Cardiovasc Res. 2009 February;81(3):592-600. PubMed PMID: 18703536, incorporated herein by referencein its entirety).

c. Anti-von Willebrand factor (VWF) antibody, purified polyclonal Ab,Goat IgG (R&D Systems, Minneapolis, Minn., USA).

d. Anti-NOS-2 antibody stain, using a commercially available kit(Novostain Super) ABC Kit (universal), NCL-ABCu (NovocastraLaboratories, Ltd., Newcastle upon Tyne, UK).

Example 4 Estimation of the Antibody Labelling in the Light MicroscopicSlides

Each specimen was stained using all the antibodies listed in Example 3.Estimation of the antibody labelling was performed usingphotomicrographs obtained via a digital microscope (Coolscope, NikonInstruments Europe BV, Amsterdam Netherlands). Ten fields were selectedrandomly from each section. All the selected fields were clear fromfreezing defects, gaps or folds. The overlap of fields was clearlyavoided. The slide selection and analysis was conducted by a blindoperator. The sections were labelled with numbers that were unknown tothe operator. The photomicrographs are shown in FIGS. 1A-1L.

Image J software was used to analyse the sections. The total labelledarea in each field was measured in pixels and expressed as percentage ofthe total field area. The mean value of the labelled area for all fieldsof a specific antibody in each group was obtained (see FIG. 1 for thephotomicrographs of all antibodies). Photomicrographs were analysedusing ImageJ software, and the % Ab-labelled-area of 10 fields perspecimen was recorded.

Example 5 Statistical Analysis Results

IBM—Statistical analysis software package—SPSS version 20 was used toanalyse the data. All data were expressed as mean±SD. Analysis ofvariance (ANOVA) was used to compare data among the groups. The level ofsignificance was set at p<0.05.

There was no significant difference in the weight of the heart (HW), theweight of the left ventricle (LVW), and the ratio of LVW/HW between thegroups. No difference were also found between the heart weight and leftventricular weight when normalized to body weight and tibial length.(Data are not shown)

FIG. 2A shows the levels of VEGF are significantly higher in theNigella-fed (2.59±1.37) and the exercise (2.51±1.86) groups compared tothe control group (1.58±0.78) (“**” indicates P<0.01). The levels ofVEGF were comparable in the Nigella-fed and the exercise-trained groups,indicating that both Nigella sativa administration and exercise trainingwere potential factors for induction of capillary growth.

FIG. 2B shows the levels of VWF are significantly lower in theNigella-fed (1.57±0.83) and the exercise (1.07±0.72) groups compared tothe control group (2.38±1.72) (“**” indicates P<0.01). This findingmight support the angiogenic effect of both long-term administration ofNigella sativa and exercise on coronary circulation. The observationalso supported the similarities in the actions of these twointerventions.

FIGS. 2C and 2D show the exercise group was found to have a higher levelof PECAM-1 (1.79±0.78) and a lower level of NOS-2 (0.83±0.57) than thecontrol group (PECAM-1: 1.19±1.17, “**” indicates P<0.01; NOS-2:1.25±1.19, “*” indicates P<0.05). The exercise protocol might be toomild to induce a significant increase in NOS-2 levels in the heart. Thetraining did not demonstrate an increase in the expression of NOS-2 inthe cross-section of the left ventricles of the exercise-trained rats.Interestingly, upregulation of NOS-2 was clearly elucidated in thehind-limb of the rats after femoral artery ligation and reperfusion.Rats fed with Nigella sativa did not show an increase in NOS-2. Nigellasativa is known to be an anti-inflammatory and antioxidant mediator andit might not culminate in overexpression of NOS-2.

The rats fed with Nigella sativa did not demonstrate a significantincrease in PECAM-1. However, the possibility of the presence of ongoingcapillary angiogenesis may still be valid because there was astatistically significant increase in the VEGF level and a statisticallysignificant decrease in the VWF level. A longer duration of Nigellaadministration might be required to achieve a higher capillary beddensity which is indicated by elevated PECAM-1 levels. By stopping theadministration of Nigella sativa during the weekends may have delayedthe onset of cardiac hypertrophy in the rats. Different stages ofcardiac and coronary adaptation to exercise training have also beenreported (Gielen S, Schuler G, Adams V. Cardiovascular effects ofexercise training: molecular mechanisms. Circulation. 2010 September;122(12):1221-38. PubMed PMID: 20855669, incorporated herein by referencein its entirety).

The disclosure demonstrated evidence of active coronary angiogenesisinduced by administration of Nigella sativa and aerobic exercisetraining in adult Wistar rats. This evidence includes a statisticallysignificant increase in the VEGF level and a reduction in the VWF level.This finding is promising in the emergence of new therapeutic andpreventive strategy for coronary artery disease in specific and otherperipheral artery diseases in general.

1: A method for effecting angiogenesis and optionally treating ischemia,the method comprising: administering a first effective amount of Nigellasativa to a subject in need thereof. 2: The method of claim 1, whereinthe method effects angiogenesis in at least one body part selected fromthe group consisting of a heart, a brain, and a limb. 3: The method ofclaim 1, wherein the method treats ischemia which is at least oneselected from the group consisting of brain ischemia, ischemic heartdisease, and limb ischemia. 4: The method of claim 3, which treats atleast one type of ischemic heart disease selected from the groupconsisting of stable angina, myocardial infarction, cardiac syndrome X,and cardiac arrest. 5: The method of claim 1, which effects angiogenesisin a heart of the subject and does not lead to cardiac hypertrophy inthe subject. 6: The method of claim 1, wherein the first effectiveamount of Nigella sativa is administered in a composition comprisingwater and Nigella sativa seeds. 7: The method of claim 6, wherein thecomposition comprises 1-50 wt % Nigella sativa seeds, based on a totalweight of the composition. 8: The method of claim 7, wherein the Nigellasativa seeds are ground. 9: The method of claim 1, wherein the firsteffective amount of Nigella sativa is administered 1-3 times a day. 10:The method of claim 1, wherein the first effective amount of Nigellasativa is administered for 6-30 weeks. 11: The method of claim 10,wherein the first effective amount of Nigella sativa is administered 4-6days per week for 7-10 weeks. 12: The method of claim 11, wherein thefirst effective amount of Nigella sativa is administered for 4-6consecutive days per week. 13: The method of claim 1, wherein the firsteffective amount of Nigella sativa is in a range of 0.1-1 g/kg bodyweight of the subject. 14: The method of claim 13, wherein the firsteffective amount of Nigella sativa is in a range of 0.6-0.9 g/kg bodyweight of the subject. 15: The method of claim 1, wherein the firsteffective amount of Nigella sativa is administered orally. 16: Themethod of claim 1, further comprising measuring a level of at least onebiomarker selected from the group consisting of VEGF, VWF, PECAM-1, andNOS-2 before and/or after the administering of the first effectiveamount. 17: The method of claim 16, further comprising: administering asecond effective amount of Nigella sativa. 18: The method of claim 17,wherein the second effective amount of Nigella sativa is in a range of0.1-1 g/kg body weight of the subject and is different from the firsteffective amount of Nigella sativa. 19: The method of claim 17, thesecond effective amount of Nigella sativa is administered for 1 day or2-10 consecutive days. 20: The method of claim 19, wherein the secondeffective amount of Nigella sativa is administered for 4-6 consecutivedays.